This R03 application aims at providing data in support of the underlying hypothesis that CD4+ T cell responses towards HIV-1 protease neoepitopes created by drug escape mutations can help to control the growth of resistant variants. A significant proportion of Pi-treated HIV-1 infected (HIV-1+) patients develop resistance mutations, and CD8+ T cell responses against such drug-induced mutant epitopes have been documented. It has been suggested that such a T cell response against epitopes under drug pressure may keep mutants under immunological control, or delay the emergence of drug resistance mutations, in patients undergoing antiretroviral (ARV) treatment. Given the importance of CD4+ T cell responses in maintaining CD8+ T cell memory, we will investigate, for the first time, the recognition of sites of Pi-induced mutations by CD4+ T cells of Pi-treated HIV-1 + patients. The study will have a cross-sectional (Aim 1) and a longitudinal arm (Aim 2). To search for CD4+ T cell responses against mutant protease epitopes (Aim 1), we will synthesize peptides representing wild-type and neoepitope sequences, incorporating major Pi-induced mutation sites and predicted to bind to multiple HLA-DR molecules in silico. To evaluate their promiscuity, we will assess their ability to bind multiple HLA-DR molecules in vitro. Subsequently, we will test the T cell responses from Pi-treated HIV-1 + patients to such peptides by IFN-gamma ELISPOT, CFSE proliferation and flow cytometry. Sequences of recognized peptides will be compared to endogenous HIV-1 protease sequence. In the longitudinal arm (Aim 2), we will study patients whose HIV-1 isolates developed new mutations in the protease gene along the 1-year observation period. Among such patients, we will compare disease evolution between the groups that developed or failed to develop CD4+ T cell responses to neoepitopes. We expect to obtain information as to whether the convergence of drug and immune pressure influences mutations leading to immune escape or drug resistance. The information to be obtained may help develop immune strategies that could be applied to enhance the immune response against mutant HIV-1 and fight the emergence of Pi-resistant HIV-1 strains.